Lignin is the the second most abundant organic polymer, exceeded only by cellulose. It binds with cellulose fibres which make up approximately one-fourth of the weight of dry wood. This aromatic polymer is recalcitrant to degradation. Lignin is covalently associated with hemicelluloses in the cell wall via numerous types of linkage. Among the linkage, the most abundant lignin substructure is the β-aryl ether, which accounts for approximately 40% of the inter phenylpropane linkages (Higuchi T 1990. Lignin biochemistry: biosynthesis and biodegradation. Wood Sci. Technol. 24: 23-63). In addition lignin does not possess any repeating units like other biopolymer such as cellulose, protein, starch etc. Ether bonds are often prevalent, providing a variety of linkages to the numerous aromatic residues. These structural features dictate constraints on the degradation of the lignin. A portion of a typical structure is illustrated in FIG. 1 and that the arylglycerol β-aryl ether structure red color in the figure is quantitatively the most important linkage, constituting at least 40% of the polymer. But, biodegradation of lignin is a prerequisite for the processing of biofuel, and wood pulp from plant raw materials. The improving of lignin degradation would drive the output from biofuel processing to better gain or better efficiency factor.
Lignin is a component of lignocellulose, which must be degraded to allow efficient use of cellulosic material for saccharification, paper production, biofuel production or upgradation of fodder. Ligincellulose degradation is a multienzymatic process involving both hydrolytic and oxidative enzymes. In general lignin composed of three principal building blocks namely, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. In addition, grass and dicot lignin also contain large amounts of phenolic acids such as p-coumaric and ferulic acid, which are esterified to alcohol groups.
Lignin peroxidases (LiP), manganese peroxidases (MnPs) and laccases are three families of enzymes that are involved in the biological degradation of lignin. LiP oxidizes nonphenolic lignin substructures by abstracting one electron and generating cation radicals (Kirk T K, Tien M, Kersten P J, Mozuch M D and Kalyanaraman B 1986. Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol β-aryl ether substructure of lignin. Biochem J 236: 279-287; Kirk T K and Farrell R L 1987. Enzymatic “combustion”: The microbial degradation of lignin. Annu Rev Microbiol 41: 465-505) and MnP oxidizes phenolic rings to phenoxyl radicals which lead to decomposition of Lignin (Gold M H, Wariishi H and Valli K 1989. Extracellular peroxidases involved in lignin degradation by the white-rot basidiomycete Phanerochaete chrysosporium. In: Biocatalysis in Agricultural Biotechnology, pp. 127-140. Edited by Whitaker J R and Sonnet P E. American Chemical Society, Washington, D.C.). Whereas laccase is capable of oxidizing both phenolic and non-phenolic moieties of lignin but that the latter is dependent on the co-presence of primary laccase substrates (Bourbonnais R and Paice M G 1990. Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett 267(1): 99-102). FIG. 2 showed a dimeric model compounds that represent the major arylglycerol β-aryl ether lignin structure undergo Cα-Cβ cleavage upon oxidation by LiP (Kirk et al. 1986. Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol β-aryl ether substructure of lignin. Biochem J 236: 279-287). Research on lignin biodegradation has increased enormously in recent years. Especially since the discovery of the first lignocellulose degrading fungus Phanerochaete chrysosporium genome was sequenced due to the interest on biological degradation of lignin (Martinez et al. 2004. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotehnol 22(6): 695-700). Perhaps the best studied, is the degradation of lignin in lignocellulose by white rot fungi, Phanerochaete chrysosporium (Aust S D 1995. Mechanisms of Degradation by White Rot Fungi. Environ Health Perspect 103:59-61; Leisola M S A, Ulmer D and Fiechter A 1984. Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium. Arch Microbiol 137: 171-175; Ulmer D, Leisola M, Puhakka J and Fiechter A 1983. Phanerochaete chrysosporium: Growth pattern and lignin degradation. Appl Microbiol Biotechnol 18: 153-157; Chua M G S, Chen C L, Chang H M and Kirk T K 1982. 13CNMR spectroscopic study of lignin degraded by Phanerochaete chrysosporium. I. New structures. Holzforschung 36: 165-172). White-rot fungi produce a range of extracellular lignolytic enzymes, including heme-dependent lignin peroxidases, manganese peroxidases, and versatile peroxidases and copper-dependent laccases (Sanchez C 2009. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol Adv 27: 185-194; Ten Have R, Teunissen P J M 2001. Oxidative mechanisms involved in lignin degradation by white-rot fungi. Chem Rev 101: 3397-3413). With the aid of these extracellular peroxidase and laccase enzyme, white-rot fungi degrade lignin (Timothy D H B, Ahmad M, Hardiman E M and Rahmanpour R 2001. Pathways for degradation of lignin in bacteria and fungi. Nat. Prod. Rep 28: 1883-1896).
Increasing interest in the exploitation of plant biomass as a renewable resource has provided an impetus for research on microbial degradation of lignocellulose. Large quantity of biomass (mainly lignocellulosic materials) is generated from forestry, agriculture and food industry which are not used in byproduct processes. To effectively utilize this plant biomass as renewable resources, alternative means of degradation of lignocellulose need to be explored. It is desirable to identify new fungi capable of degrading lignin for use in the manufacture of cellulosic products from lignocellulosic materials.
In order to be efficient, the degradation of lignin requires several types of enzymes acting cooperatively. At least two categories of enzymes are necessary to degrade lignin: Lignin peroxidase that oxidizes nonphenolic lignin substructures and laccase that oxidize both phenolic and nonphenolic lignin substructure thus they can degrade cooperatively. To realize and commercialize the mentioned enzymes, stable supply of various lignin degrading proteins are needed. Therefore, it is desirable for the industry to completely identify these lignin degrading genes and their encoded proteins, thus utilizing the genetic information for degradation of lignin from lignocellulosic materials.